This invention relates to cell cycle regulation. More specifically, this invention relates to novel ubiquitin carrier polypeptides (Ubc""s) involved in the ubiquitination and degradation of cyclins, and to nucleic acid encoding these proteins. This invention also relates to inhibitors of such Ubc""s and to kits for and methods of screening for compounds which inhibit the ubiquitination, and hence the destruction, of cyclins.
Mitotic entry and exit in most organisms is controlled by the synthesis and destruction of cyclin B, a positive regulatory subunit of the protein kinase Cdc2, the catalytic component of mitosis promoting factor (MPF) (Norbury et al. (1992) Ann. Rev. Biochem. 61:441-470; Murray (1995) Cell 81:149-152). Cyclins are marked for destruction by the covalent addition of ubiquitin at the end of mitosis (Glotzer et al. (1991) Nature 349:132-138; Hershko et al. (1991) J. Biol. Chem. 266:16376-16379; Hershko et al. (1994) J. Biol. Chem. 269:4940-4946). Ubiquitinated cyclins are then rapidly degraded by the 26S proteasome (Hershko et al. (1994) J. Biol. Chem. 269:4940-4946). This process is catalyzed by a cyclin-specific ubiquitin ligase, E3-C, which is part of a 20S particle, the cyclosome (Sudakin et al (1995) Mol. Biol. Cell. 6:185-198). Cyclosome activation is initiated by Cdc2 (Fxc3xa9lix et al. (1990) Nature 346:379-382; Sudakin et al. (1995) Mol. Biol. Cell. 6:185-198) and terminated by an okadaic acid-sensitive phosphatase (Lahav-Baratz et al. (1995) Proc. Nat. Acad. Sci. USA, in press). This particle contains homologs of two yeast proteins, Cdc16 and Cdc27 (King et al. (1995) Cell 81:279-288), proteins required for the destruction of cyclin B and the metaphase-anaphase transition (Tugendreich et al. (1995) Cell 81:261-268; Irniger et al (1995) Cell 81:269-277).
Cyclosome-associated E3-C catalyzes cyclin ubiquitination using a specialized ubiquitin conjugating enzyme or carrier protein (E2); also called Ubc, originally identified in clam as E2-C (Hershko et al. (1994) J. Biol. Chem. 269:4940-4946). Multiple species of E2""s were first found in animal cells (Pickart et al (1985) J. Biol. Chem. 260:1573-1581), and at least ten different Ubc""s have now been identified in yeast (Jentsch (1992) Ann. Rev. Genetics 26:179-207).
Structurally, all known E2""s share a conserved domain of approximately 16 kD. This domain contains the cysteine (Cys) residue required for the formation of ubiquitin-E2 thiol ester. Certain E2 enzymes contain additional typical domains. Based on their structure, the E2 enzymes can be divided into three groups (Jentsch (1992) Ann. Rev. Genet. 26:179-207)). Class I E2""s consist almost exclusively of the conserved domain. Class II proteins have C-terminal extensions that may contribute to substrate recognition or to cellular localization. For example, yeast Ubc2 and Ubc3 have a highly acidic C-terminal domain that promote interaction with basic substrates such as histones (Jentsch (1992) Ann. Rev. Genet. 26:179-207)). Class III enzymes have various N-terminal extensions; however, their function is not known.
Genetic and molecular analysis has revealed that different Ubc""s have different cellular functions. Two closely related Ubc""s, Ubc4 and Ubc5, appear responsible for ubiquitin-dependent degradation of most short-lived and abnormal proteins (Jentsch (1992) Ann. Rev. Genetics 26:179-207). Ubc2 (RAD6) is required for several functions, including DNA repair, sporulation (Sung et al. (1988) Genes and Dev. 2:1476-1485) and N-end rule degradation (Dohmen et al (1991) Proc. Natl. Acad. Sci. USA 88:7351-7355). Ubc3 (Cdc34) is required for the G1/S transition (Goebl et al. (1988) Science 241:1331-1335), where it appears to participate in the ubiquitin-dependent destruction of the G1 cyclin dependent kinase (cdk) inhibitor, p40sic1 (Schwob et al (1994) Cell 79:233-244). Ubc9 is required for cell cycle progression in late G2 or early M; both CLB5, an S phase cyclin, and CLB2, an M phase cyclin, are stable in Ubc9 mutants, suggesting that Ubc9 may be responsible for cyclin ubiquitination (Seufert et al (1995) Nature 373:78-81). E2-C, a clam Ubc was determined to be one of the components of the clam oocyte system responsible for the specific ubiquitination of cyclin (Hershko et al. (1994) J. Biol. Chem. 269:4940-4946).
However, heretofore, the Ubc(s) responsible for the ubiquitination of the mitotic cyclins in humans were unidentified and characterized.
It has been discovered that both clam and human have novel cyclin-selective ubiquitin carrier polypeptides which are involved in the ubiquitination of proteins and ubiquitin-directed protein degradation. These findings have been exploited to develop the present invention, which is directed to human and clam ubiquitin carrier polypeptides and inhibitors thereof, to nucleic acids encoding such polypeptides, and to methods employing such ubiquitin carrier polypeptides and inhibitors.
In a first aspect, the invention provides an isolated and purified, non-xenopal, ubiquitin carrier polypeptide (Ubc) involved in the ubiquitination of cyclin A and/or B.
As used herein, the term xe2x80x9cisolated and purifiedxe2x80x9d refers to polypeptides which are substantially free of contaminating cellular or other associated components, including, but not limited to proteinaceous, carbohydrate, or lipid impurities. This term is also meant to encompass molecules which are homogeneous by one or more purity or homogeneity characteristics used by those with skill in the art. For example, an isolated and purified Ubc will show constant and reproducible characteristics within standard experimental deviations for parameters such as molecular weight, chromatographic migration, amino acid composition, HPLC profile, biological activity, and other such parameters. The term is not meant to exclude artificial and synthetic mixtures of the Ubc with other compounds.
The term xe2x80x9cnon-xenopalxe2x80x9d refers to Ubc""s which are not derived from frog cells or encoded by frog nucleic acid.
As used herein, the term xe2x80x9cinvolved inxe2x80x9d means xe2x80x9cwhich takes part inxe2x80x9d and is meant to encompass the role played or function that a Ubc has during ubiquitination of cyclin A and/or B. This role includes an enzymatic activity required for transporting ubiquitin to cyclin A or B. The xe2x80x9cUbc-specific N-terminal extensionxe2x80x9d referred to in this aspect of the invention is used to describe a unique (outside of the conserved domain) amino acid sequence of at least 5, or preferably, at least 10, more preferably, at least 15, more preferably at least 20, more preferably, at least 25, most preferably between 30-32 amino acid residues having sequence homology to the unique amino acid sequence(s) found in clam E2-C, human UbcH10, and frog Ubc-x.
In some embodiments, the Ubc is recombinantly produced. In other embodiments, fragments of the Ubc are provided which are enzymatically active and demonstrate the same or substantially similar ubiquitin carrier polypeptide function as the full length Ubc. As used herein a xe2x80x9cfragmentxe2x80x9d of a molecule such as E2-C, UbcH10, or inhibitors thereof, refers to any smaller polypeptide subset of that molecule. In some embodiments, the Ubc is a clam or human Ubc. In some embodiments, the Ubc has an amino acid sequence with about 61-100%, more preferably, about 75-100%, and most preferably with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:1 or 3. By xe2x80x9chomologyxe2x80x9d is meant sequence identity or similarity.
By similarity is meant the degree to which amino acid changes are in accordance with the conservative amino acid substitutions exemplified in Table 1 below.
In particular embodiments, the Ubc has the amino acid sequence set forth as SEQ ID NO:1 or 3. In yet other embodiments, the polypeptide is encoded by a nucleic acid hybridizable with a second nucleic acid set forth as SEQ ID NO:2 or 4. Preferably, the polypeptide is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid having SEQ ID NO:2 or 4. Stringent hybridization conditions are known by those with skill in the art (see, e.g., Ausebel et al., Protocols in Molecular Biology, John Wiley and Sons, Inc., New York, N.Y. (1989): hybridization in 50% formamide, high salt (either 5xc3x97SSC (20xc3x97: 3 M NaCl/0.3 M trisodium citrate) or 5xc3x97SSPE (20xc3x97: 3.6 M NaCl/0.2 M NaH2PO4/0.02 M EDTA, pH 7.7)), 5xc3x97Denhardt""s solution, and 1% SDS) at low stringency: room temperature; moderate stringency: 42xc2x0 C.; and high stringency: 68xc2x0 C.
In some embodiments, the N-terminal extension has about 61-100% homology, preferably 75-100%, and more preferably has about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:9 or 10. In particular embodiments, the N-terminal extension has the amino acid sequence set forth as SEQ ID NO:9 or 10. In yet other embodiments, the N-terminal extension is encoded by a nucleic acid hybridizable, preferably under stringent conditions, with a second nucleic acid encoding the amino acid sequence set forth as SEQ ID NO:9 or 10.
In another aspect, the invention provides a nucleic acid encoding the Ubc""s, and fragments thereof, of the invention as described above. In some embodiments, the nucleic acid is a cDNA, and in particular embodiments, the cDNA has the nucleotide sequence set forth as SEQ ID NO:2 or 4. In some embodiments, the nucleic acid of the invention encodes a human Ubc having an amino acid sequence with about 61-100% homology, preferably about 74-100%, and more preferably, with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:1. In other embodiments the nucleic acid of the invention encodes a clam Ubc having an amino acid sequence with about 61-100%, preferably with about 75-100%, and more preferably, with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:3. Also provided is a nucleic acid hybridizable under stringent conditions with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:2 or 4.
In another aspect, the present invention provides a selective inhibitor of Ubc polypeptide function. As used herein, the term xe2x80x9cUbc functionxe2x80x9d is meant to encompass the enzymatic transfer of ubiquitin from E1 to E2 and from E2 to a protein target, e.g., cyclin A or B. xe2x80x9cUbc functionxe2x80x9d also refers to the association of E2 and E3. The term xe2x80x9cinhibitors of Ubc functionxe2x80x9d is meant to include agents that block the transfer of ubiquitin from E1 to E2 and agents that block the transfer of ubiquitin from E2 to a protein target, e.g., cyclin A or B. As used herein, xe2x80x9cinhibitors of Ubc functionxe2x80x9d is also meant to include agents that block association between E2 and E3. All such agents prevent cyclin ubiquitination. It is preferred that the agent be a selective inhibitor of Ubc function, more preferably wherein the Ubc is selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof. Suitable assays for measuring Ubc function according to the present invention include those which allow measurement of the formation of E-2-ubiquitin thiol ester, measurement of the formation of ubiquitin- or multi-ubiquitin-conjugates of a cyclin, or measurement of cyclin degradation. Assays that allow measurement of cell cycle progression may also be used according to the present invention.
The agents screened in the above-described assay methods can be, but are not limited to peptides, polypeptides, antibodies, carbohydrates, vitamin derivatives, or other pharmaceutical agents. These agents can be selected and screened 1) at random, 2) by a rational selection, or 3) by design using, for example, protein or ligand modeling techniques.
For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to or block the activity of the Ubc. Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be xe2x80x9crationally selected or designedxe2x80x9d when the agent is chosen based on the configuration of the above-described Ubc or known ligand.
The present invention further relates to selective inhibitors of Ubc function or cyclin ubiquitination identified by the above-described screening and assay methods, which can include peptides, polypeptides, antibodies, carbohydrates, vitamin derivatives, or other pharmaceutical agents. In one embodiment, the inhibitor is a dominant negative mutant of a ubiquitin carrier protein, or a fragment thereof capable of inhibiting Ubc function. As described hereinabove and in the exemplification below, a mutant of UbcH10 containing a cysteine serine mutation at residue 114 is as a dominant negative mutant. The dominant negative mutant overcomes the activity of wild type UbcH10 and inhibits cyclin ubiquitination and degradation.
As used herein, a xe2x80x9cselective inhibitorxe2x80x9d is a compound which preferentially interferes with Ubc function. Preferably, the selective inhibitor reduces the enzymatic function of the novel Ubc""s of the invention. In some embodiments, the inhibitor is a dominant negative mutant. As used herein, a xe2x80x9cdominant negative mutantxe2x80x9d is a polypeptide variant of a wild type Ubc with which it competes or interferes for its ubiquitin carrier function. Dominant negative mutants of the novel Ubc""s of the invention inhibit cell cycle progression, blocking both the destruction of mitotic cyclins A and B, and the onset of anaphase. In some embodiments, the dominant negative mutant is recombinantly produced. In other embodiments, dominant negative mutants of the invention have a serine-residue in place of a cysteine residue in a conserved region of the polypeptide. In specific embodiments, the dominant negative mutant of the invention comprises a serine residue at position 114 substituted for a cysteine residue. In some embodiments, the dominant negative mutant inhibits the function of a human or clam Ubc. The dominant negative mutant has an amino acid sequence with about 61-100%, preferably about 75-100%, and more preferably, about 94-100%, homology to the amino acid sequence set forth as SEQ ID NO:5 or 7 in some embodiments. In other embodiments, the dominant negative mutant is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6 or 8. In yet other embodiments, the invention provides a fragment of the dominant negative mutant which inhibits Ubc function.
The invention also provides a nucleic acid encoding the dominant negative mutant described herein. In some embodiments, the nucleic acid is hybridizable under stringent conditions with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6 or 8. The nucleic acid may be a cDNA which, in some embodiments, has the nucleotide sequence set forth as SEQ ID NO:6 or 8. In other embodiments, the nucleic acid of the invention encodes a dominant negative mutant having an amino acid sequence with about 61-100% homology, preferably about 75-100%, and more preferably, with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:5 or 7.
Kits useful for the ubiquitination and degradation of a cyclin are also provided by the invention. These kits include (a) a ubiquitin-human ubiquitin carrier polypeptide complex, wherein the ubiquitin carrier polypeptide is an isolated and purified, non-xenopal, Ubc involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension. In preferred embodiments, the Ubc is clam E2-C, human UbcH10, or an enzymatically active fragment of clam E2-C or UbcH10; and (b) a ubiquitin ligase (E3).
In some embodiments, the cyclin to be degraded is cyclin A or cyclin B and the ubiquitin-ubiquitin carrier polypeptide complex comprises human UbcH10 having an amino acid sequence set forth as SEQ ID NO:1. In another embodiment, the cyclin to be degraded is cyclin A or cyclin B and the ubiquitin-ubiquitin carrier polypeptide complex comprises clam E2-C having an amino acid sequence set forth as SEQ ID NO:3. In some embodiments, the ubiquitin-ubiquitin carrier protein complex comprises a Ubc having an amino acid sequence with about 61-100%, preferably about 75-100%, and more preferably, about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:1 or 3. In particular embodiments, the Ubc in the complex has the amino acid sequence set forth as SEQ ID NO:1 or 3. In yet other embodiments, the Ubc in the complex is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid set forth as SEQ ID NO:2 or 4. In some embodiments, the Ubc has an N-terminal extension which has about 61-100%, preferably about 75-100%, and more preferably about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:9 or 10. In particular embodiments, the Ubc in the complex has an N-terminal extension with an amino acid sequence set forth as SEQ ID NO:9 or 10.
In another aspect, the invention provides other kits useful for the ubiquitination and degradation of a cyclin including ubiquitin, a ubiquitin activating enzyme (E1), ATP, a ubiquitin carrier protein selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof, and a ubiquitin ligase (E3). In some embodiments, the cyclin to be degraded is cyclin A or cyclin B and the ubiquitin-ubiquitin carrier protein complex comprises human UbcH10 having an amino acid sequence set forth as SEQ ID NO:1. In other embodiments, the cyclin to be degraded is cyclin A and/or cyclin B and the ubiquitin-ubiquitin carrier protein complex comprises clam E2-C having an amino acid sequence set forth as SEQ ID NO:3.
The invention also provides a method of ubiquitinating a cyclin and/or targeting a cyclin for degradation, comprising the step of contacting the cyclin with a ubiquitin-ubiquitin carrier protein complex, the ubiquitin carrier polypeptide being an isolated and purified non-xenopal Ubc involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension; and a ubiquitin ligase (E3). In preferred embodiments, the Ubc is selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof. In some embodiments, the ubiquitin-ubiquitin carrier protein complex comprises a Ubc having an amino acid sequence with about 61-100%, preferably about 75-100%, and more preferably, with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:1 or 3. In particular embodiments, the Ubc in the complex has the amino acid sequence set forth as SEQ ID NO:1 or 3. In yet other embodiments, the Ubc in the complex is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid set forth as SEQ ID NO:2 or 4. In some embodiments, the Ubc has an N-terminal extension which has about 61-100% and more preferably, about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:9 or 10. In particular embodiments, the Ubc in the complex has an N-terminal extension with an amino acid sequence set forth as SEQ ID NO:9 or 10.
A method of inhibiting Ubc function is also provided by the invention. In one embodiment, an inhibitor of a Ubc is administered to the cell in an amount sufficient to inhibit the Ubc function, e.g., by inhibiting the ubiquitination of a cyclin. In preferred embodiments, the inhibitor is a dominant negative mutant according to the invention and as described above. In some embodiments, the Ubc is a mutant clam E2-C. In other embodiments, the Ubc is a mutant human UbcH10. In some embodiments, the dominant negative mutant is recombinantly produced. In specific embodiments, the dominant negative mutant of the invention comprises a serine residue at position 114 substituted for a cysteine residue. In some embodiments, the dominant negative mutant inhibits the function of a human or clam Ubc. The dominant negative mutant has an amino acid sequence with about 61-100%, more preferably, about 75-100%, and most preferably, about 94-100%, homology to the amino acid sequence set forth as SEQ ID NO:5 or 7 in some embodiments. In other embodiments, the dominant negative mutant is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6 or 8. In yet other embodiments, the invention provides a fragment of the dominant negative mutant which inhibits Ubc function. In one preferred embodiment, the method of inhibiting Ubc function results in the inhibition of cell proliferation.
The present invention further relates to a method of screening for compounds which inhibit Ubc function. In this method an assay is provided for measuring Ubc function, wherein the assay comprises a ubiquitin carrier polypeptide selected from the group consisting of a non-xenopal ubiquitin carrier polypeptide involved in the ubiquitination of cyclin a and/or B and having a Ubc-specific N-terminal extension and an enzymatically active fragment thereof. The assay is performed in the presence and absence of a compound to-be-tested. The amount of change in Ubc function measured in the presence of the compound as compared to Ubc function measured in the absence of the compound is then determined, a reduction of Ubc function measured in the presence of the compound indicating that the compound is an inhibitor of Ubc function. In preferred embodiments, the ubiquitin carrier polypeptide is selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof. More preferably, the ubiquitin carrier polypeptide is isolated and purified.
In another aspect, the invention provides a method of screening for compounds which inhibit the ubiquitination of cyclins. In this method, ubiquitin, a ubiquitin activating enzyme (E1), ATP, an isolated and purified, non-xenopal, Ubc involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension, a ubiquitin ligase (E3), Cdc2, and a cyclin are incubated in the presence and in the absence of a compound to be tested. The amount of cyclin-ubiquitin-Cdc2 complex formed in the presence and absence of the compound is then measured, a reduction in the amount of complex formed in the presence of the compound indicating that the compound is an inhibitor of cyclin ubiquitination. As used herein, the term xe2x80x9ccyclin-ubiquitin-Cdc2 complexxe2x80x9d refers to ubiquitin covalently bound to cyclin B complexed to Cdc2.
In preferred embodiments, the Ubc is selected from the group consisting of clam E2-C, human UbcH10, or an enzymatically active portion thereof. Preferably, the ubiquitin carrier polypeptide is isolated and purified. In some embodiments, the human UbcH10 or clam E2-C has an amino acid sequence with about 61-100%, preferably about 75-100%, and more preferably, with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:1 or 3, respectively. In particular embodiments, UbcH10 and E2-C have the amino acid sequences set forth as SEQ ID NO:1 and 3, respectively. In yet other embodiments, UbcH10 and E2-C are encoded by a nucleic acid hybridizable under stringent conditions, with a second nucleic acid set forth as SEQ ID NO:2 and 4, respectively. In some embodiments, UbcH10 has an N-terminal extension which has about 61-100%, preferably about 75-100%, and more preferably about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:9, and E2-C has an N-terminal extension which has about 61-100%, preferably about 75-100%, and more preferably, about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:10. In particular embodiments, the N-terminal extension of UbcH10 and E2-C has the amino acid sequence set forth as SEQ ID NO:9 and 10, respectively.
Also provided by the invention are antibodies specific for E2-C and for UbcH10, and antisense oligonucleotides specific for E2-C or UbcH10 nucleic acids.
In yet another aspect, the invention provides therapeutic formulations comprising a selective inhibitor of ubiquitin carrier protein function in an amount sufficient to inhibit the ubiquitination of a cyclin, and a pharmaceutically acceptable carrier. In preferred embodiments, the inhibitor comprises a dominant negative mutant of a ubiquitin carrier protein, or a fragment thereof capable of inhibiting Ubc function. In some embodiments, the dominant negative mutant has a serine residue at position 114 substituted for a cysteine residue. In particular embodiments, the dominant negative mutant has an amino acid sequence which is at least about 90-95% homologous with the amino acid sequence set forth as SEQ ID NO:5 or 7. In other embodiments, the dominant negative mutant is encoded by a nucleic acid which is hybridizable under stringent conditions with the nucleic acid having a nucleotide sequence set forth as SEQ ID NO:6 or 8.